Escherichia coli O157: H7 Igikoresho cyo Kumenya Acide Nucleic (PCR Fluorescent Probe Method)
Ibisobanuro:
Injangwe.FP102
Ikoreshwa mugushakisha byihuse no gusuzuma E. coli O157: H7 mubiribwa, ibiryo, ibyitegererezo byamazi hamwe nibidukikije.
Ibisobanuro
Ikoreshwa mugushakisha byihuse no gusuzuma E. coli O157: H7 mubiribwa, ibiryo, ibyitegererezo byamazi hamwe nibidukikije.
[Ihame ry'ikizamini]Ukurikije ihame rya tekinoroji ya fluorescent PCR, primers yihariye na Taqman probe zakozwe kuri gene yihariye ya Escherichia coli O157: H7, kandi igaragazwa nigikoresho cya fluorescent PCR, kugirango tumenye neza Escherichia coli O157: H7 Kumenya neza ADN.
Ibirimo
Icyitonderwa: Umuyoboro wa ROX ntabwo urimo.
| CIbigize | Ibisobanuro | Quantity |
| Buffer A. | Itiyo | 1 |
| Buffer B. | Itiyo | 1 |
| Kugenzura neza | Itiyo | 1 |
| Kugenzura nabi | Itiyo | 1 |
Biteganijwe Gukoreshwa
Byakoreshejwe Kuri gutahura byihuse no gusuzuma E. coli O157: H7 mubiribwa, ibiryo, urugero rwamazi hamwe nibidukikije.
Imiterere yo kubika nigihe cyo kurangiriraho
Bika kuri -20 ℃ mu mwijima kandi wirinde gukonjesha no gukonja.
Igihe cyemewe ni 12amezi, kandi itariki yo kubyara irerekanwa kumupaki yo hanze.
Ibikoresho n'ibikoreshwa
Fluorescent igereranya PCR igikoresho, imbunda ya pipette ninama zihuye, vortex shaker, mini centrifuge.
Ikoreshwa
1. Icyitegererezo
1.1 Ubwoko bw'icyitegererezo: Iki gikoresho kibereye ibiryo, ibiryo, icyitegererezo cy'amazi hamwe nizindi ngero zikekwa ko zanduye na Escherichia coli O157: H7.Ku bicuruzwa bitunganijwe cyane, ibinyobwa nibindi bintu birimo pigment, bigomba kwozwa kugirango birinde kugira ingaruka ku ikusanyamakuru rya fluorescence.
1.2 Gutunganya icyitegererezo: Reba kuri "GB 4789.10-2016 Umutekano wibiryo byigihugu Ibiribwa bisanzwe Microbiologiya Ikizamini cya Escherichia coli O157: H7 Ikizamini" kugirango hategurwe icyitegererezo, umuco wo gukungahaza no kwigunga kwa Escherichia coli O157: H7.
- Ngukuramo aside ucleic
Fata mL 20 yumuti ukungahaye muri 1.5 mL ya centrifuge, ongeramo 200 μL ya lysate ya mikorobe (ibikoresho byongeweho birakenewe), vortex kumasegonda 30, centrifuge mugihe gito, hanyuma ushire kuruhande.
Icyitonderwa: Gukuramo aside nucleic muri lysate bigomba kurangira muminota 10, kandi ntibishobora kubikwa igihe kirekire.
3. Nucleic Acide Amplification
3.1 Fungura igikoresho cya fluorescent PCR ikoreshwa.
Buffer A na Buffer B kuva mubikoresho, kubishonga neza, na centrifuge mugihe gito.Ongeramo 18 μL Buffer A na 2 μL Buffer B kuri buri cyuma cya PCR.Noneho ongeramo 5 mL buriwese kugenzura nabi, gukuramo acide nucleic, no kugenzura neza mumiyoboro ya PCR, kanda igituba, na centrifuge mugihe gito.
3.3 Kwimura umuyoboro wa PCR kumashini ya fluorescent PCR, hanyuma ukoreshe inzira zikurikira kugirango ukore igeragezwa rya amplification: hitamo 25 mL kuri sisitemu yo kubyitwaramo, gukusanya ibimenyetso bya fluorescence kuri 60 ° C kuri buri cyiciro, hanyuma uhitemo FAM kumuyoboro wo gutahura.
| Intambwe | Gahunda | Umubare wizunguruka |
| 1 | 37 ℃ 5min | 1 |
| 2 | 9 5 ℃ 3min | 1 |
| 3 | 95 ° C 15s | 4 0 |
| 60 ℃ 30s (gukusanya fluorescence) |
Imiyoboro yo gusesengura ibibazo
The following is an analysis of the problems that might be encountered in the extraction of viral RNA. We wish it would be helpful to your experiment. In addition, for other experimental or technical problems other than operating instructions and problem analysis, we have dedicated technical support to help you. Contact us if you need at : 028-83361257or E-mail：Tech@foregene.com。
Nta RNA ishobora gukururwa cyangwa umusaruro wa acide nucleic ni muke
Mubisanzwe hariho ibintu byinshi bigira ingaruka kumikorere yo gukira, nka: icyitegererezo cya RNA, uburyo bwo gukora, ingano ya elution, nibindi .。
Isesengura ry'impamvu zisanzwe ：
1.Kora ubwogero cyangwa ubushyuhe buke (4 ° C) centrifugation mugihe ukora.
Icyifuzo: Ubushyuhe bwicyumba (15-25 ° C) imikorere, ntuzigere woga urubura hamwe na centrifuge yubushyuhe buke.
2. Kubika icyitegererezo kidakwiye cyangwa ububiko bw'icyitegererezo igihe kirekire.
Icyifuzo: Bika ingero kuri -80 ° C cyangwa uhagarike muri azote yuzuye, kandi wirinde gukoresha inshuro nyinshi;gerageza gukoresha ingero zegeranijwe zivuye muri RNA.
3.Icyitegererezo kidahagije
Icyifuzo: Nyamuneka menya neza ko icyitegererezo nigisubizo cyakazi (Linear Acrylamide) byavanze neza kandi bigashyirwa muminota 10 mubushyuhe bwicyumba (15-25 ° C)
4.Umuhanga wongeyeho nabi
Icyifuzo: Menya neza ko ddH2O yubusa ya RNase yongewe hagati ya membrane yinkingi yo kweza
5.Ubunini budakwiye bwa anhydrous ethanol muri Buffer viRW2
Igitekerezo: Nyamuneka kurikiza amabwiriza, ongeramo ingano yukuri ya anhydrous ethanol kuri Buffer viRW2 hanyuma ubivange neza mbere yo gukoresha ibikoresho。
6.Ikoreshwa ry'icyitegererezo kidakwiye.
Igitekerezo: 200µl yicyitegererezo kuri 500μl ya Buffer viRL.Ingano yikitegererezo irenze izagabanya igipimo cyo gukuramo RNA.
7.Ijwi ridakwiriye cyangwa gusohora kutuzuye.
Igitekerezo: Ingano nziza yinkingi yo kweza ni 30-50μl;niba ingaruka zo gukuraho zidashimishije, birasabwa kongeramo ubushyuhe bwa RNase-Ubusa ddH2O hanyuma wongere umwanya ushira mubushyuhe bwicyumba, nka 5-10min
8.Inkingi yo kweza ifite ibisigisigi bya Ethanol nyuma yo koza muri Buffer viRW2.
Igitekerezo: Niba Ethanol ikomeje kuguma nyuma yo koza muri Buffer viRW2 hamwe na centrifugation yubusa ya 2min, inkingi yo kweza irashobora gusigara mubushyuhe bwicyumba cya 5min nyuma ya centrifugation yubusa kugirango ikureho Ethanol isigaye.
Kwangirika kwa molekile ya RNA isukuye
Ubwiza bwa RNA yatunganijwe bujyanye nibintu nko kubika icyitegererezo, kwanduza RNase, no gukora.
Isesengura ry'impamvu zisanzwe ：
1.Icyitegererezo cyegeranijwe nticyabitswe mugihe.
Igitekerezo: Niba icyitegererezo kidakoreshejwe mugihe cyo gukusanya, nyamuneka ubike kuri -80 ℃ cyangwa azote yuzuye.Kubikuramo molekile ya RNA, gerageza gukoresha ingero zegeranijwe igihe cyose bishoboka.
2.Ingero zegeranijwe zarakonje kandi zishonga inshuro nyinshi.
Igitekerezo: Irinde gukonjesha no gukonjesha inshuro nyinshi (bitarenze rimwe) mugihe cyo gukusanya no kubika, bitabaye ibyo umusaruro wa acide nucleique uzagabanuka.
3.RNase yatangijwe mucyumba cyo gukoreramo cyangwa nta gants imwe ikoreshwa, masike, nibindi byambarwa.
Igitekerezo: Gukuramo igeragezwa rya molekile ya RNA bikorwa neza mubyumba bitandukanye bya RNA, kandi imbonerahamwe yubushakashatsi isukurwa mbere yubushakashatsi.Wambare uturindantoki hamwe na mask mugihe cyo kugerageza kugirango wirinde kwangirika kwa RNA guterwa na RNase.
4.Ragent yandujwe na RNase mugihe cyo kuyikoresha.
Icyifuzo: Simbuza Virus RNA nshya yo kwigunga kubushakashatsi bujyanye.
5.Indwara ya RNase yanduye ya centrifuge, inama za pipeti, nibindi. Icyifuzo: Menya neza ko imiyoboro ya centrifuge, inama za pipeti, hamwe na pipeti byose ari RNase-Free.
Molekile ya RNA yatunganijwe yagize ingaruka kubushakashatsi bwo hasi
Molekile ya RNA yatunganijwe ninkingi yo kweza izagira ingaruka kubigeragezo byo hepfo niba hari ion nyinshi cyangwa umunyu mwinshi, nka: transcription transcription, Blot y'Amajyaruguru, nibindi .。
1.Hariho ion z'umunyu zisigaye muri molekile ya RNA yatowe.
Icyifuzo: Menya neza ko ingano yukuri ya etanol ya anhydrous yongerewe kuri Buffer viRW2, hanyuma ukarabe inkingi yo kweza kabiri ukurikije umuvuduko ukwiye wa centrifugation kumabwiriza yimikorere ； Niba hakiri ioni yumunyu usigaye, urashobora kongeramo Buffer viRW2 kumurongo wogusukura, hanyuma ukayirekera mubushyuhe bwicyumba cya 5min.Noneho kora centrifugation kugirango ukureho umunyu ion wanduye kurwego runini
2.Hariho Ethanol isigaye muri molekile ya RNA yatowe
Igitekerezo: umaze kwemeza ko inkingi zo kwezwa zogejwe na Buffer viRW2, kora centrifugation yubusa-yubusa ukurikije umuvuduko wa centrifugal kumabwiriza yimikorere.Niba hasigaye etanol isigaye, irashobora gusigara muminota 5 mubushyuhe bwicyumba nyuma ya centrifugation yubusa kugirango ikureho Ethanol isigaye kurwego runini.
Imfashanyigisho:
Igitabo gikubiyemo amabwiriza ya virusi ya RNA
